Hemoglobin (Hb) E (2E26K) is the most common worldwide naturally occurring mutant Hb with a mutation at the 1121 interface. EE individuals exhibit a mild, chronic anemia while HbE/2-thalassemia individuals show a range of clinical manifestations, including high morbidity, and death, often resulting from cardiac dysfunction. The significant role of HbE in the red blood cell pathophysiology and molecular mechanisms giving rise to the HbE diseases is enigmatic. Since, HbE has been shown to have normal oxygen affinity (Bunn et al., 1972), we have proposed a possible mechanism whereby HbE may have reduced capacity to generate sufficient bioactive nitric oxide (NO) (1) to confer protection against high levels of membrane damaging reactive oxygen species (ROS) arising from the 2-thalassemia and (2) as a secondary NO source for endothelial functioning. In support of this hypothesis we have obtained preliminary data from HbE showing decreased nitrite reductase activity compared to HbA. Our group recently obtained the high resolution deoxy and liganded HbE structures (Protein Data Bank entries 1YVQ, 1YVT, 3DUT) and found that the tertiary conformations within the T and R quaternary structures of HbE are altered relative to HbA. The proposed project which builds on these two findings, seeks to establish the extent and molecular origins of the altered nitrite reactivity. The project will utilize innovative sol-gel encapsulation protocols to trap and characterize the reactivity of the T and R state of HbE with respect to a series of reactions proposed to contribute to the production of bioactive NO. The project seeks to determine whether the source of HbE altered reactivity to generate bioactive forms of NO from nitrite arises from changes in allostery, in local tertiary structure or in the redox properties of the T and R states. These studies are of significance in that they are likely to provide a novel mechanism for the origin of HbE-derived pathophysiology with implications for other Hb related pathologies and diseases linked to endothelial dysfunction. There is the potential for a new paradigm in which to develop therapies for HbE 2-thalassemia. PUBLIC HEALTH RELEVANCE: This proposal aims to understand the fundamental causes that give rise to Hemoglobin E diseases, one of which can cause body and mental retardation and can lead to death, usually from heart malfunction. It is the aim of this proposal to test the hypothesis that this disease originates from properties of HbE that reduce its capacity to produce nitric oxide from nitrite. These findings have the potential to change the way doctors view and treat the often fatal HbE/2-thalassemia disease.